HISTONE/DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

染色质生物合成过程中的组蛋白/DNA 相互作用

• 批准号: 2430467
• 负责人: Anthony Annunziato
• 金额: $ 22.65万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430467
• 关键词: DNA replication; HeLa cells; acetylation; acyltransferase; chemical association; chromatin; enzyme inhibitors; histones; immunoprecipitation; nucleosomes; phosphorylation; protein biosynthesis

DESCRIPTION (Adapted from Applicant's Abstract): The long-term objective of this research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the propagation of epigenetic information to progeny cells. To this end, the present proposal focuses on the function of histone acetylation and phosphorylation during chromatin replication, and on the analysis of nucleosome pre-assembly complexes that have been observed in HeLa, a transformed human cell line. Experiments will be performed to dissect the composition of soluble non- nucleosomal histone complexes that accumulate when DNA replication is inhibited, and to identify possible somatic histone escort proteins. This will be accomplished by 1) immunoprecipitating radiolabeled cell extracts with anti-histone antibodies, including antibodies specific for acetylated H4; and 2) probing the purified complexes with antibodies that recognize known somatic nucleosome assembly factors. The involvement of H4 acetylation in escort protein recognition will also be assessed, by challenging somatic cell extract with synthetic polypeptides that represent either the acetylated or the unacetylated N-terminus of histone H4. In parallel experiments, the ability of soluble histone complexes to participate in nucleosome assembly in vivo and in vitro will also be determined. As part of the above studies, the properties of a cytosolic histone acetyltransferase, which preliminary experiments strongly suggest is involved in acetylation of newly synthesized histone H4, will be examined. The activity, specificity, and cell cycle regulation of this acetyltransferase (which is specific for H4, and appears to be a member of the "acetyltransferase B" family) will be determined through a straightforward in vitro assay, using either purified histones or acetylated and unacetylated H4 N-terminal peptides as substrates. The ability of this acetyltransferase to facilitate histone complex formation will also be examined. Finally, antibodies that recognize the phosphorylated, but not the unphosphorylated forms of histone H1 will be used to study the role of H1 phosphorylation in chromatin replication. Immunoprecipitations of cross- linked (radiolabeled) chromatin fragments will be performed to determine the distribution of phosphorylated H1 with respect to newly replicated DNA, and to newly assembled nucleosomes. By immunoprecipitating chromatin DNA labeled during replication in cycloheximide, the relative degree to which phosphorylated H1 is presented on segregated parental nucleosomes will also be analyzed.

描述（改编自申请人摘要）：长期目标 这项研究计划的目的是阐明体细胞的机制， 染色质生物合成，并了解表观遗传的传播 信息传递给后代细胞。 为此目的，本建议的重点是 对组蛋白乙酰化和磷酸化的作用 染色质复制和核小体预组装分析 在一种转化的人类细胞HeLa中观察到的复合物 线 将进行实验以剖析可溶性非- 核小体组蛋白复合物的积累时，DNA复制是 抑制，并确定可能的体细胞组蛋白护送蛋白。 这将通过1）免疫沉淀放射性标记的细胞 抗组蛋白抗体的提取物，包括对 乙酰化的H4;和2）用抗体探测纯化的复合物 识别已知的体细胞核小体组装因子。 的 参与护送蛋白识别H4乙酰化也将 通过用合成的 代表乙酰化或未乙酰化的多肽 组蛋白H4的N末端。 在平行实验中， 可溶性组蛋白复合物参与体内核小体组装 也将在体外进行测定。 作为上述研究的一部分， 乙酰转移酶，初步实验强烈表明， 参与新合成的组蛋白H4的乙酰化， 考察 这种蛋白的活性、特异性和细胞周期调控 乙酰基转移酶（对H4特异，似乎是一个成员， “乙酰转移酶B”家族中的一个）将通过 直接的体外测定，使用纯化的组蛋白或 乙酰化和未乙酰化的H4 N-末端肽作为底物。 的 这种乙酰转移酶促进组蛋白复合物的能力 培训也将被审查。 最后，抗体识别磷酸化的蛋白质，但不识别磷酸化的蛋白质。 非磷酸化形式的组蛋白H1将被用于研究的作用， 染色质复制中的H1磷酸化。 免疫沉淀 将进行交联（放射性标记）染色质片段， 确定磷酸化H1相对于新的 复制的DNA和新组装的核小体。 通过 免疫沉淀染色质DNA标记在复制过程中， 环己酰亚胺，磷酸化H1的相对程度， 还将分析分离的亲本核小体上呈现的蛋白质。